P132	Homeobox A9 regulates the angiogenic function of endothelial cells and endothelial progenitor cells: role of integrin expression.
C.Urbich, T.Brühl, A.M.Zeiher, S.Dimmeler
Molecular Cardiology, University of Frankfurt, Frankfurt/Main, DE.

Homeobox proteins are transcriptional regulators of the cardiovascular system during development. In the adult organism, homeobox A9 (HoxA9) modulates angiogenic functions and acts as an endothelial regulatory factor. As we have recently shown, ischemia-induced blood vessel formation is significantly impaired in HoxA9-/- mice as compared to wild-type (wt) mice. Since defective angiogenesis or vasculogenesis might be the cause of impaired neovascularization capacity of HoxA9-deficient mice, we investigated the contribution of HoxA9 for both endothelial cell (EC) and endothelial progenitor cell (EPC) function and integrin expression as potential target genes known to be involved in angiogenesis. Knock-down of HoxA9 by RNA interference (RNAi) in HUVEC significantly decreased integrin αv (36±12% control, p<0.05, n=4) and integrin ß3 protein expression (55±13% control, p<0.05, n=4). In contrast, HoxA9 antisense transfection did not affect integrin α5 or integrin ß1 expression. Overexpression of HoxA9 significantly enhanced integrin αv expression (αv: 163±21% control, p<0.05, n=4). To determine a direct regulatory role of HoxA9 for integrin αvß3, we investigated the binding of HoxA9 to the integrin αv or ß3 promoters by chromatin-immunoprecipitation. Endogenous HoxA9 did not bind to the integrin αv or ß3 promoter, suggesting an indirect regulatory mechanism. To assess EC and EPC function, we used a co-culture matrigel assay of mouse aortic EC and splenic EPC isolated from HoxA9-deficient mice. Tube formation was significantly reduced in HoxA9+/- ECs (39.3±6% of wt, p<0.01, n=3) and HoxA9-/- ECs (7.0±0.9% of wt, p<0.01, n=3) as compared to wild-type cells. Moreover, tube formation was also significantly reduced in co-cultures of HoxA9+/- ECs with HoxA9+/- EPCs (52.7±5.2% of wt, p<0.01, n=6) and HoxA9-/- ECs with HoxA9-/- EPCs (13.7±1.7% of wt, p<0.01, n=6) as compared to co-cultured wild-type cells. Taken together, HoxA9 regulates tube forming activity of endothelial cells and endothelial progenitor cells and integrin αvß3 expression, and, thereby, significantly contributes to the modulation of endothelial cell function.

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